Molecular Biology of the Cell
Vol. 13, 1144 –1157, April 2002
Reconstitution and Characterization of Budding Yeast
␥-Tubulin Complex
Dani B.N. Vinh,* Joshua W. Kern,* William O. Hancock,†
Jonathon Howard,‡§ and Trisha N. Davis*㥋
*Departments of Biochemistry and ‡Physiology and Biophysics, University of Washington, Seattle,
Washington 98195; and †Department of Bioengineering, Pennsylvania State University, University
Park, Pennsylvania 16802
Submitted December 24, 2001; Accepted January 14, 2002
Monitoring Editor: Tim Stearns
Nucleation of microtubules is central to assembly of the mitotic spindle, which is required for each
cell division. ␥-Tubulin is a universal component essential for microtubule nucleation from
centrosomes. To elucidate the mechanism of microtubule nucleation in budding yeast we reconstituted and characterized the yeast ␥-tubulin complex (Tub4p complex) produced in insect cells.
The recombinant complex has the same sedimentation coefficient (11.6 S) as the native complex in
yeast cell extracts and contains one molecule of Spc97p, one molecule of Spc98p, and two
molecules of Tub4p. The reconstituted Tub4p complex binds preformed microtubules and has a
low nucleating activity, allowing us to begin a detailed analysis of conditions that enhance this
nucleating activity. We tested whether binding of the recombinant Tub4p complex to the spindle
pole body docking protein Spc110p affects its nucleating activity. The solubility of recombinant
Spc110p in insect cells is improved by coexpression with yeast calmodulin (Cmd1p). The
Spc110p/Cmd1p complex has a small sedimentation coefficient (4.2 S) and a large Stokes radius
(14.3 nm), indicative of an elongated structure. The Tub4p complex binds Spc110p/Cmd1p via
Spc98p and the Kd for binding is 150 nM. The low nucleation activity of the Tub4p complex is not
enhanced when it is bound to Spc110p/Cmd1p, suggesting that it requires additional components
or modifications to achieve robust activity. Finally, we report the identification of a large 22 S
Tub4p complex in yeast extract that contains multimers of Spc97p similar to ␥-tubulin ring
complexes found in higher eukaryotic cells.
INTRODUCTION
The microtubule cytoskeleton plays essential roles in chromosome segregation, cellular organization, and vesicle trafficking in eukaryotic cells. Effective activities of the microtubule network require precise spatial and temporal control
of assembly. In most cells, the centrosome is the primary
microtubule-organizing center that nucleates and organizes
microtubules. ␥-Tubulin is a conserved protein that localizes
to all centrosomes and is required for microtubule nucleation (Oakley et al., 1990; Horio et al., 1991; Stearns et al.,
1991; Zheng et al., 1991; Joshi et al., 1992). The majority of
cytoplasmic ␥-tubulin is found in soluble complexes with a
ring-like structure (␥-tubulin ring complex, ␥-TuRC) as examined by electron microscopy (Zheng et al., 1995; Oegema
et al., 1999; Murphy et al., 2001). Ring-like structures labeled
DOI: 10.1091/mbc.02– 01– 0607.
§
Present address: Max Planck Institute of Molecular Cell Biology
and Genetics, 10307 Dresden, Germany.
㛳
Corresponding author. E-mail address: [email protected].
1144
with ␥-tubulin are also observed in the pericentriolar material of centrosomes (Moritz et al., 1995; Vogel et al., 1997),
suggesting a role for the ␥-TuRC in microtubule nucleation
from centrosomes.
In Saccharomyces cerevisiae, ␥-tubulin or Tub4p exists in a
stable complex with Spc97p and Spc98p (Knop et al., 1997;
Knop and Schiebel, 1997). The ␥-tubulin small complex (␥TuSC) in higher eukaryotes contains orthologs of Tub4p,
Spc97p, and Spc98p and forms the core subunit in the
␥-TuRC (Martin et al., 1998; Murphy et al., 1998; Tassin et al.,
1998; Oegema et al., 1999). Vertebrate ␥-TuRCs have sedimentation coefficients ranging from 25 to 32 S (Stearns and
Kirschner, 1994; Meads and Schroer, 1995; Moritz et al., 1998;
Murphy et al., 1998, 2001; Oegema et al., 1999). In addition to
multimers of ␥-TuSC, at least five other components are
found in each ␥-TuRC (Fava et al., 1999; Gunawardane et al.,
2000; Zhang et al., 2000; Murphy et al., 2001). These additional components are required for assembly of the ␥-TuRC
and/or recruitment of the ␥-TuRC to the centrosomes. Recent electron microscopic tomography reconstruction studies propose a structural model for the Drosophila ␥-TuRC in
© 2002 by The American Society for Cell Biology
Yeast ␥-Tubulin Complex
which a ring of repeating V-shaped units of ␥-TuSCs are
capped at one end with the remaining components (Moritz
et al., 2000).
In vitro, ␥-TuRCs can promote microtubule polymerization and cap microtubule minus ends (Zheng et al., 1995;
Oegema et al., 1999; Wiese and Zheng, 2000). However,
␥-tubulin alone has also been found to bind, cap, and promote microtubule polymerization (Li and Joshi, 1995; Leguy
et al., 2000). Two models are currently proposed for the
mechanism of microtubule nucleation (reviewed in Erickson, 2000). In the template model, the ␥-TuRC acts as a
template with a ring of 13 ␥-tubulins on which the ␣/␤
protofilaments grow longitudinally and the microtubule end
becomes flushed with the ␥-TuRC surface. In the protofilament model, a single molecule or short filament of ␥-tubulins stacked longitudinally to each other, unwinds from the
␥-TuRC and acts as the seed from which ␣/␤ protofilaments
grow both laterally and longitudinally. Recent electron microscopic studies provide evidence for the template model
to explain how the ␥-TuRC is structurally incorporated into
the microtubule. Microtubules polymerized in the presence
of ␥-TuRCs or derived from animal centrosomes and yeast
spindle pole bodies contain pointed ends that are capped
similarly to the structure of purified ␥-TuRC as analyzed by
electron microscopic tomography (Byers et al., 1978; Moritz
et al., 2000). Consistent with these findings, components of
the ␥-TuRC are also localized to the capped ends of microtubules (Keating and Borisy, 2000; Zhang et al., 2000). However, the biochemical mechanism of nucleation remains unclear.
In S. cerevisiae, microtubules are only found to emanate
from the spindle pole body (SPB), which is the sole microtubule organizing center. The substructures of the SPB include the outer plaque, which organizes cytoplasmic microtubules; the central plaque, which is embedded in the
nuclear envelope; and the inner plaque, which organizes
nuclear microtubules. All three components of the Tub4p
complex (Tub4p, Spc97p, and Spc98p) are localized to the
outer and inner plaques and play key roles in microtubule
nucleation and spindle assembly (Rout and Kilmartin, 1990;
Sobel and Snyder, 1995; Marschall et al., 1996; Spang et al.,
1996a; Knop et al., 1997). Yeast exhibits a closed mitosis in
which the nuclear membrane remains intact throughout the
cell cycle, resulting in independent regulation of cytoplasmic and nuclear microtubule polymerization. This simpler
regulatory network together with the extremely well-defined SPB offers many advantages for the studies of microtubule nucleation in budding yeast. A combination of genetic and two-hybrid analyses shows that the Tub4p
complex is anchored to the inner plaque by interaction with
Spc110p (Knop and Schiebel, 1997; Nguyen et al., 1998).
Spc110p is a major SPB protein that has a calmodulin binding domain at the C terminus embedded in the SPB core, and
a Tub4p complex binding domain at the N terminus pointed
toward the microtubule ends (Geiser et al., 1993; Kilmartin et
al., 1993; Kilmartin and Goh, 1996; Spang et al., 1996b; Sundberg et al., 1996). The two domains are separated by a coiled
coil rod that acts as a spacer between the central and inner
plaques (Kilmartin et al., 1993). The human ortholog of
Spc110p is kendrin, an alternative splice variant of pericentrin that contains a C-terminal extension, including a calmodulin-binding region (Flory et al., 2000). Pericentrin has
Vol. 13, April 2002
previously been shown to play a role in recruiting the
␥-TuRC to centrosomes (Dictenberg et al., 1998), suggesting
that the mechanism of assembling the nucleation machinery
at the centrosome may be conserved between yeast and
human.
Centrosome-dependent microtubule polymerization in
higher eukaryotes involves recruitment of the ␥-TuRC to the
centrosome and activation of the nucleation machinery that
results in aster formation (Felix et al., 1994; Stearns and
Kirschner, 1994; Moritz et al., 1998; Schnackenberg et al.,
1998). Studies in Drosophila indicate that although the large
␥-TuRC has robust nucleating activity in vitro, the ␥-TuSC
has weak activity (Oegema et al., 1999; Gunawardane et al.,
2000). The fact that a ␥-TuRC–like complex has not been
found in yeast raised the question as to whether the Tub4p
complex has nucleating activity. To address these questions,
we have reconstituted the Tub4p complex and measured its
in vitro microtubule nucleating activity. Because microtubules are only found at the SPB and Spc110p is the only
component known to directly link the Tub4p complex to the
nuclear side of the SPB, we further postulate that anchoring
the Tub4p complex to the SPB may regulate its nucleating
activity. We therefore characterized the assembly and function of the Tub4p complex when bound to Spc110p. Our
results indicate that linking to Spc110p does not enhance the
nucleation activity of the Tub4p complex. This result led to
a careful examination of Tub4p complexes in yeast extracts
and identification of a large complex that may be the yeast
version of the higher eukaryotic ␥-TuRC.
MATERIALS AND METHODS
Baculovirus Construction
Recombinant proteins were produced using the Bac-to-Bac baculovirus/insect cell expression system (Invitrogen, Carlsbad, CA).
Yeast genes were inserted into the baculovirus vectors pFastbac or
pFastbacDual (Table 1). Clones of SPC97, SPC98, TUB4, SPC110, or
CMD1 were derived from plasmids previously described (Nguyen
et al., 1998). Plasmids carrying SPC29 and SPC42 were from S.
Francis (University of Washington, Seattle, WA). A plasmid carrying tub4-34 was a gift from R. Jeng and T. Stearns (Stanford University, Palo Alto, CA).
Antibodies
Anti-Spc98p antibodies were generated against a glutathione Stransferase (GST)-Spc98p fusion protein containing residues 551–
846 of Spc98p. The fusion protein was expressed in Escherichia coli,
enriched on the basis of its insolubility in 6 M urea, and gel purified
before injection into chickens (Aves Labs, Tigard, OR). Anti-Spc97p
antibodies were produced in chickens by using a 10XHis-Spc97p
(full-length) fusion protein expressed in insect cells. The insoluble
fusion protein was enriched on the basis of its insolubility in 250
mM KCl and gel purified. Anti-PY antibodies were generated from
tissue culture supernatant of mouse monoclonal cell line AK1310
(gift of R. Deshaies, California Institute of Technology, Pasadena,
CA). Anti-Spc110p and anti-Cmd1p antibodies have been described
elsewhere (Geiser et al., 1991, 1993). Anti-Tub4p was a gift of T.
Stearns (Stanford University), monoclonal anti-FLAG was a gift of
G. Zhu (University of Washington, Seattle, WA), and rabbit polyclonal anti-GST, mouse monoclonal anti-MYC, and mouse monoclonal anti-hemagglutinin (HA) antibodies were from Santa Cruz
Biotechnology (Santa Cruz, CA).
1145
D.B.N. Vinh et al.
Table 1. Expression vectors
Protein
Cmd1p
Spc29p-Flag
Spc42p-2PY
Spc97p
Spc97p-GST
10XHis-Spc97p
Spc98p
GST-Spc98p551–846
Spc110p-GST
GST-Spc110p/Cmd1p
GST-Spc110p1–220
Tub4p
Tub4-34p
Parent vector
Method of construction
pFastBac
pFastBac
pFastBac
pFastBac
pFastBac
pFBNHis10c
pFastBac
pGEX2T
pFastBac
pFastBac Dual
pMIT77c
pFastBac
pFastBac
Subclone
PCR with epitope taga
PCR with epitope taga
Subclone
PCR and subcloneb
Subclone
Subclone
Subclone
PCR and subcloneb
Subclone and PCRd
PCRe
Subclone
Subclone
a
Gene was amplified by PCR with primers encoding the epitope
tag.
b
GST was first subcloned into pFastBac to make pFastBac-GST. The
3⬘ end of the yeast gene was amplified using primers that removed
the stop codon and added a PstI site. The PCR fragment was
subcloned into the pFastBac-GST vector at the PstI site. The remainder of the gene was then added.
c
The vector pFBNHis10 is pFastBac containing a 10⫻His tag at the
N terminus, and pMIT77 is pFastBac containing an N-terminal GST
(both were gifts of K. Kaplan and P. Sorger, Massachusetts Institute
of Technology, MA).
d
Both CMD1 and SPC110 were cloned into pFastBacDual. GST was
generated by PCR and inserted at the 5⬘ end of SPC110.
e
Nucleotides 1– 660 of the SPC110 open reading frame were amplified using a 3⬘ primer encoding a stop codon.
Baculovirus Expression
We used Sf9 or Hi5 insect cell lines for baculovirus expression.
Recombinant baculoviruses were produced from Sf9 cells as recommended by the manufacturer’s instructions (Invitrogen). For recombinant proteins, Hi5 cells grown on plates were used for small-scale
studies (5–10 ⫻ 106 cells), and Sf9 cells grown in shaking flasks were
used for large purification preps (⬎2 ⫻ 108 cells). Cells were coinfected with the appropriate baculoviruses for 48 –72 h. The amount
of virus was optimized for protein expression. Infected cells were
harvested and spun down at 500 ⫻ g for 10 min. Cell pellets were
gently washed once with phosphate-buffered saline, repelleted, and
then either processed for protein purification or frozen in liquid
nitrogen and stored at ⫺80°C.
Protein Purification and GST-Copurification Assay
We purified the Tub4p complex by using two methods. In method
1, Tub4p and Spc98p were copurified with Spc97p-GST from cells
coinfected with the three viruses and the GST domain subsequently
removed by cleavage. Two to three volumes of HB100 (40 mM
K-HEPES, pH 7.5, 100 mM KCl, 1 mM EGTA, 1 mM MgCl2, 0.1 mM
GTP) ⫹ 1 mM dithiothreitol (DTT) ⫹ 1 mM phenylmethylsulfonyl
fluoride (PMSF) ⫹ 10 ␮g/ml pepstatin A ⫹ a 1:1000 dilution of a
stock of three protease inhibitors (10 mg/ml each of chymostatin,
leupeptin, and aprotinin [CLA]) were added to frozen cell pellets
from 1 liter of infected culture. The pellets were then quickly
thawed at room temperature (RT) and the cell suspension was
sonicated twice with a tip sonicator. NP-40 (1% vol/vol) was then
added to the suspension and incubated on ice for 30 – 45 min. The
lysate was centrifuged at 40,000 ⫻ g for 40 min in a Beckman Coulter
JA25.50 rotor, and the cleared supernatant loaded onto a column (2
1146
ml) of glutathione-Sepharose (Amersham Biosciences, Piscataway,
NJ) equilibrated in HB100. The column was washed with at least 20
column volumes of HB100 ⫹ 1 mM DTT, but minus protease
inhibitors. The Spc97p/Spc98p/Tub4p complex was released by
incubation for 4 h at 4°C with GST-PreScission protease (Amersham
Biosciences) suspended in HB100 ⫹ 1 mM DTT. Protease cleavage
results in the addition of six amino acid residues (LEVLFQ) after the
last codon of Spc97p. Samples were eluted in HB100 ⫹ 1 mM DTT
⫹ 1/10,000 dilution of CLA, and fractions containing protein were
identified by dot blotting on nitrocellulose. Peak fractions were
pooled and concentrated with Microcon or Centricon filters (Millipore, Bedford, MA). Sucrose was added to a final concentration of
10% (wt/vol), the protein solution was frozen in liquid nitrogen and
stored at ⫺80°.
In method 2, Spc97p, Spc98p, and Tub4p were copurified with
GST-Spc110p1–220 from cells coinfected with the four viruses. The
large complex [GST-Spc110p1–220 ⫹ Tub4p complex] was first affinity purified with glutathione-Sepharose beads as described above.
Then the entire large complex was eluted with 10 mM glutathione in
25 mM Tris buffer, pH 7.8, 50 mM KCl, 1 mM MgCl2. Fractions were
pooled and applied to a 1.7-ml POROS Q/M anion exchange column run on the BioCAD Sprint Perfusion Chromatography System
(Applied Biosystems, Foster City, CA) to separate GST-Spc110p1–220
from the Tub4p complex. Samples were eluted with a 0 –1 M KCl
gradient in 25 mM Tris buffer, pH 7.8, 1 mM MgCl2 and analyzed by
SDS-PAGE. Peak fractions were pooled, concentrated, and dialyzed
into HB100 ⫹ 10% glycerol ⫹ 1 mM DTT ⫹ 1 ⫻ 10⫺5 dilution of
CLA protease inhibitors, followed by freezing in liquid nitrogen and
storage at ⫺80°C. The usual yield of method 1 is 900 ␮g of Tub4p
complex per 1 liter of infected cell culture, whereas method 2 can
produce up to 3 mg of Tub4p complex per 1 liter of culture. Protein
concentration was determined either using bicinchoninic acid (Sigma Chemical, St. Louis, MO), or by comparison with known
amounts of bovine serum albumin (BSA) after separation on SDSPAGE and staining with Coomassie blue. Protein bands were
scanned using a Wratten filter (#0.2) on an ArcusII, Agfa scanner.
NIH Image software was used to quantify band intensities.
To purify an Spc110p-GST/Cmd1p complex, frozen cell pellets
were quickly lysed at RT in HB300 (HB100, except with 300 mM
KCl) plus 4 mM DTT, 1 mM PMSF, 20 ␮g/ml pepstatin, and a 1/500
dilution of CLA. NP-40 was added to a final concentration of 1%
and the sample incubated on ice for 30 min. The lysate was initially
spun at 40,000 ⫻ g for 40 min, and then the supernatant was respun
at 80,000 ⫻ g for 10 min in a Sorvall F28/36 rotor. The cleared
supernatant was loaded onto a glutathione-Sepharose column and
washed with five column volumes of HB300, five column volumes
of HB200, and at least 10 column volumes of HB100. Samples were
either eluted with glutathione (producing Spc110p-GST/Cmd1p) or
by protease treatment (producing Spc110p/Cmd1p) as described
above.
In the GST-copurification assay, 5 ⫻ 106 infected cells were processed per sample. Different combinations of baculoviruses were
coinfected with a virus expressing GST-tagged protein and cell
lysates prepared in HB100 and spun at 14,000 ⫻ g in a microfuge.
Incubation of the cleared supernatant with glutathione beads and
elution with glutathione in HB100 were both done in microfuge
tubes on a nutator for 30 min each at 4°C. The eluted samples were
analyzed by Western blot to look for their copurification with the
GST-tagged protein.
Estimating Kd for Spc110p Binding to Tub4p
Complex
A fixed concentration of 138 nM purified Tub4p complex was incubated at RT for 30 min with various concentrations of purified
Spc110p-GST/Cmd1p in a constant volume of HB100 containing
10% glycerol, 2 mM DTT, 0.1% NP-40, and 0.3% casamino acids.
Each sample was then added to an equal volume of glutathioneSepharose beads equilibrated in HB100 with 2 mM DTT, 0.1%
Molecular Biology of the Cell
Yeast ␥-Tubulin Complex
NP-40, and 0.3% casamino acids, and incubated for another 20 min
at RT. Samples were centrifuged for 1 min at 2000 ⫻ g; the supernatant was carefully removed and analyzed by SDS-PAGE. The
relative amount of unbound Tub4p complex in the supernatant was
determined by quantifying the intensity of the Tub4p bands on a
Coomassie-blue stained gel. The fraction of unbound [Tub4p complex] shown in Figure 5 ⫽ (intensity of unbound Tub4p at a given
[Spc110p]/intensity of unbound Tub4p in the absence of Spc110p).
The binding equation for Spc110p and Tub4p complex is as
follows:
Kd ⫽
关free Spc110p兴 ⫻ 关free Tub4p兴
关Spc110p 䡠 Tub4p兴
where [free Spc110p] ⫽ [total Spc110p] ⫺ [Spc110p 䡠 Tub4p], and
[Spc110p 䡠 Tub4p] ⫽ [total Tub4p] ⫺ [free Tub4p]. By solving for
[free Tub4p] as a function of [total Spc110p] and [total Tub4p] and
using linear regression (SigmaPlot) to fit the data, we estimated the
Kd for binding to be 150 nM.
Sucrose Gradient Sedimentation and Gel Filtration
Chromatography
Sedimentation coefficients of proteins were determined by sucrose
gradient centrifugation. To generate a larger complex containing
Spc110p/Cmd1p and the Tub4p complex, 200 nM purified Spc110p
was incubated with 700 nM purified Tub4p complex for 30 min at
RT before loading on the gradient. Sucrose gradients (10 – 40%) were
generated by allowing five steps of equal volume to diffuse into
continuous gradients at RT and chilled at 4°C before use. Gradient
buffers were either HB100 or TB100 (see “Yeast Extract” below) with
1 mM DTT, 0.1 mM PMSF, 1 ␮g/ml pepstatin A, and a 1 ⫻ 10⫺4
dilution of CLA. Each experimental run was calibrated against
protein standards that were loaded onto the same gradient as the
tested sample. Protein standards BSA (4.4 S), aldolase (7.4 S), catalase (11.3 S), and thyroglobulin (19.4 S) (all from Sigma Chemical)
were dissolved in either HB100 or TB100. Between 100 and 200 ␮g
of total protein was loaded onto each gradient and centrifuged at
210,000 ⫻ g for 5 h at 4°C in a TLS55 rotor. Thirteen to fourteen
fractions (150 ␮l) were collected from the top with cut off pipette
tips. Recombinant proteins and standards were analyzed directly by
SDS-PAGE followed by staining with Coomassie blue. When yeast
extracts were analyzed, protein fractions were first precipitated with
10% trichloroacetic acid (TCA) and then analyzed by Western blot.
Protein gel bands were quantified and peak fractions identified.
The Stokes radii of protein complexes were estimated by gel
filtration chromatography on a Superose-6 column (Amersham Biosciences) by using the Biocad sprint perfusion chromatography
system. Each experimental run was calibrated against standards:
dextran (void volume), thyroglobulin (8.6 nm), ferritin (6.1 nm),
catalase (5.2 nm), aldolase (4.6 nm), and BSA (3.5 nm). The column
buffer was HB100 containing 1 mM DTT and 0.1 mM GTP. Peak
fractions (300 ␮l) identified by absorbance at 230 nm were precipitated with 10% TCA and analyzed by Western blots. The molecular
mass of each protein complex was estimated using the method of
Siegel and Monty (1966).
Nucleation Assay
Reagents. BRB80 is 80 mM K-piperazine-N,N⬘-bis(2-ethanesulfonic
acid) buffer, pH 6.9, 1 mM EGTA, and 1 mM MgCl2; GTP stock
(Roche Applied Sciences, Indianapolis, IN) is 100 mM in 100 mM
MgCl2; HB100gly is HB100 containing 10% glycerol. All assay buffers were made at one time, distributed to small aliquots, frozen, and
stored at ⫺20°C. The background microtubule polymerization activity was similar when the same buffer was assayed within a year
of storage. Tubulin was purified from bovine brain (Mitchison and
Kirschner, 1984) and labeled with tetramethylrhodamine as described (Hyman et al., 1991).
Vol. 13, April 2002
Assay. The nucleation assay was optimized using short rhodamine
microtubule seeds (⬍2 ␮m) generated by polymerizing microtubules in the presence of guanylyl-(␣,␤)-methylene-diphosphonate
(Hyman et al., 1991). The concentration of rhodamine tubulin used
in the assay was chosen because it elongated ⬎80% of guanylyl(␣,␤)-methylene-diphosphonate seeds in 4 min and gave the lowest
background of polymerization in the absence of seeds. A 5-␮l total
reaction included 2.5 ␮l of HB100gly alone or purified yeast proteins
diluted in HB100gly, plus a 2.5-␮l solution of 2.0 mg/ml rhodamine
tubulin, 1 mM GTP, and 1⫻ BRB80 (from a 5⫻ stock). Polymerization was achieved by incubation for 4 min at 37°C, and stopped by
adding 50 ␮l of prewarmed 1% gluteraldehyde (Ted Pella, Redding,
CA) in BRB80. Samples were gently mixed with a cut off pipette tip,
incubated for 3 min at RT, and then 1 ml of ice-cold BRB80 was
added to each sample, the tubes gently inverted, and transferred to
ice. Various microtubule dilutions (50 ␮l) were each pelleted in
TLS55 centrifuge tubes at 173,000 ⫻ g for 8 min, 4°C, through 0.5 ml
of BRB80 and a cushion of 0.75 ml of 15% glycerol in BRB80 onto
poly-lysine– coated 5-mm coverslips placed on Teflon adaptors. After centrifugation, coverslips were carefully removed, postfixed in
methanol at ⫺20°C, briefly air-dried, mounted on slides with Citifluor (Ted Pella), and sealed with nail polish. Rhodamine microtubules were viewed with a 100⫻ objective on a fluorescence microscope (Diastar, Leica, NY). Magnified images were projected onto a
silicon-intensified target camera (Hamamatsu C2400-8, Bartels and
Stout, WA), displayed on a high-resolution video monitor, and
recorded on VHS tapes. Microtubules were counted in 20 – 40 random video fields, and specific images were captured on the computer by using NIH Image software.
Microtubule Copelleting Assay
Taxol-stabilized microtubules were made by incubating bovine tubulin in 1⫻ BRB80 plus 10% dimethyl sulfoxide, 1 mM GTP, and 1
mM DTT for 30 min at 37°C, and stopping the reaction with 9
volumes of 20 ␮M taxol (Sigma Chemical) in BRB80. The solution
was cleared by centrifugation in a microfuge at 5000 ⫻ g to remove
aggregates. The remaining microtubules were collected by pelleting
through a 33% glycerol/taxol/DTT/BRB80 cushion in a TLA100-2
rotor for 20 min at 200,000 ⫻ g, RT. The pellet was rinsed once and
resuspended in 10 ␮M taxol in BRB80 ⫹ 1 mM DTT. Tubulin
concentration was quantified using the Bradford assay (Bio-Rad
reagent; Bio-Rad, Hercules, CA).
For the copelleting assay, all tested samples were subjected to a
prespin of 120,000 ⫻ g for 2 min, at 2°C in a TLA45 rotor. Various
concentrations of taxol stabilized microtubules were incubated for
30 min at RT with either 10 ␮M taxol/DTT/BRB80 buffer, purified
Tub4p complex, or purified bacterial GST in the presence of 0.5
mg/ml BSA prepared in taxol/BRB80. The sample was then pelleted through a 15% glycerol/taxol/BRB80 cushion at 109,000 ⫻ g
for 10 min in a TLA100 rotor at 25°C. The supernatant was removed
from the microtubule pellet and then both supernatant and pellet
were analyzed by Western blotting.
Yeast Strains, Cell Extracts, and
Immunoprecipitation
SPC97 was tagged in either CRY1 (MATa) or CRY2 (MAT␣), which
are isogenic yeast strains with the genotype ade2–1oc can1-100 his311,15 leu2-3,112 trp1-1 ura3-1. We used the polymerase chain reaction (PCR)-based gene targeting method originally described in
Wach et al. (1997) and as modified in http://depts.washington.edu/
⬃yeastrc/fm_home3.htm. pFA6a-13MYC-kanMX6 was used as the
PCR template to create SPC97-13MYC cells (DVY52) and pFA6a3HA-kanMX6 was used to create SPC97–3HA cells (DVY51) (Bahler
et al., 1998). To make the SPC97-13MYC/SPC97-3HA diploid
(DVY53), DVY52 (MAT␣), and DVY51 (MATa) cells were mated to
each other and the resulting diploids tested for their ability to
sporulate. Heterozygous SPC97-13MYC/SPC97 (DVY54) and
1147
D.B.N. Vinh et al.
SPC97-3HA/SPC97 (DVY55) strains were generated by crossing
DVY52 or DVY51 to CRY1 or CRY2, respectively. In all cases, correct
protein expression was confirmed by Western blots with anti-MYC
and/or anti-HA antibodies. All yeast strains appear to grow normally on plates and in culture at 30°C.
To make cell extracts, all strains were grown in YPD at 30°C to a
density of 2 ⫻ 107 cells/ml. Cells were pelleted and washed once
with water, stored at ⫺80°C, or resuspended in the appropriate lysis
buffer. For extracts in TB100 (20 mM Tris buffer, pH 7.5, 100 mM
NaCl, 10 mM EDTA, 2 mM EGTA), buffer and lysis conditions of a
40-ml culture cell pellet were essentially as described in Knop et al.
(1997), except 5% glycerol was omitted in the buffer and the final
protease inhibitors included 1 mM PMSF and a 1/1000 dilution of
CLA. An extract in HB100 buffer was made by first resuspending a
cell pellet from a 40-ml culture in 500 ␮l of HB (HB100 without KCl)
plus 1 mM EDTA, 2 mM DTT, 1 mM PMSF, and 1/1000 dilution of
CLA. Cells were vortexed with glass beads until ⬎90% of cells were
lysed. Cell lysate was retrieved to a fresh tube, and an additional 250
␮l of HB was added to the glass beads. The entire sample was spun
for 10 s in a microfuge to remove cell debris, and then for another 20
min at 13,000 ⫻ g, 4°C. The pellet was resuspended in 100 –200 ␮l of
HB100 plus 1 mM EDTA, 2 mM DTT, 1 mM PMSF, 1/1000 dilution
CLA, and 1% NP-40 and incubated for 30 min on ice. Insoluble
material was removed by centrifugation at 100,000 ⫻ g for 15 min at
4°C. The final supernatant was saved for sucrose gradient centrifugation or immunoprecipitation. Protein concentrations were measured using bicinchoninic acid.
To immunoprecipitate Spc97p-13MYC, 4 ␮g of anti-MYC antibodies was added per 100 ␮g of cell extract and incubated at 4°C for 1 h,
followed with another hour of incubation in protein-G Sepharose
(Amersham Biosciences) at 4°C. Similar protocols were used for
immunoprecipitating Spc97p-3HA, except 8 ␮g of anti-HA antibodies was used per 100 ␮g of cell extract. Precipitates on beads were
pelleted at 2000 ⫻ g for 1 min and washed three times with HB100
containing 1 mM DTT, 1/1000 dilution of CLA, and 0.1% NP-40.
Precipitates were resuspended in 2⫻ sample buffer and analyzed by
Western blots.
RESULTS
Interactions of Tub4p Complex with Spc110p
Previous studies by Knop et al. (1997) indicated that Tub4p
exists predominantly in complex with Spc97p and Spc98p in
yeast cells. We and others have subsequently shown that
these three components are anchored to the nuclear side of
the SPB via interactions with the core component Spc110p
(Knop and Schiebel, 1997; Nguyen et al., 1998). Genetic analyses and two-hybrid assays indicated that the interactions
between Spc98p and the N terminus of Spc110p are direct
and robust, whereas Spc97p and Tub4p depend on Spc98p
for their interactions with Spc110p (Nguyen et al., 1998). To
better understand these diverse interactions, we tested the
ability of components of the Tub4p complex to bind directly
to the first 220 residues of Spc110p fused to GST. We infected insect cells with different combinations of recombinant baculoviruses and tested which components copurify
with GST-Spc110p1–220. Initial experiments showed that coexpression of Tub4p greatly increases the production of
soluble Spc98p and soluble Spc97p (Figure 1A), so we included Tub4p in all subsequent experiments. Tub4p itself
associates very poorly with GST-Spc110p1–220 (Figure 1B).
When Tub4p, Spc98p, and Spc97p are expressed in the starting supernatant, all three proteins copurified with GSTSpc110p1–220 (Figure 1C). In the absence of Spc98p, a barely
detectable amount of Spc97p and a low level of Tub4p
1148
Figure 1. Spc98p binds most robustly to the N-terminal domain of
Spc110p. Insect cells were infected with different combinations of
viruses as indicated. (A) Production of soluble Spc97p and Spc98p is
greatly enhanced upon coexpression with wild-type Tub4p. Western blots show the cleared lysates derived from insect cells expressing Spc98p or Spc98p and Spc97p with and without the coexpression of Tub4p or Tub4 –34p. (B) Tub4p interacts poorly with GST or
GST-Spc110p1–220. Tub4p was tested for its ability to bind glutathione-Sepharose beads, GST, or GST-Spc110p1–220. The Western blot
shows the starting cleared lysates (S) (0.25% of total) and the eluates
(E) (5% of total) from the glutathione-Sepharose beads. (C) An
Spc98p/Tub4p complex binds GST-Spc110p1–220 as efficiently as an
Spc98p/Tub4p/Spc97p complex. Herein, we loaded 0.5% of S and
10% of E. Antibodies used in all Westerns include anti-Spc97p,
anti-Spc98p, anti-Tub4p, and anti-GST.
copurify with GST-Spc110p1–220. In contrast, the expression
of only Spc98p and Tub4p allows efficient copurification of
both components with GST-Spc110p1–220. We conclude that
Spc98p is the primary component interacting with Spc110p.
Molecular Biology of the Cell
Yeast ␥-Tubulin Complex
Figure 2. Two methods of generating purified
Tub4p complex reconstituted from insect cells. (A)
Method 1: the Tub4p complex was isolated by copurification with Spc97p-GST followed by cleavage
of GST. (B) Method 2: the Tub4p complex was copurified with GST-Spc110p1–220. Numbers indicate
positions of molecular weight standards. In both
methods, a similar stoichiometry of 1 Spc98p:1
Spc97p:2 Tub4p was observed. (C) GST-Spc1101–220
is purified away from the Tub4p complex by anion
exchange chromatography in method 2. Numbers
above panel indicate fractions collected from the
anion exchange column. (A–C) SDS-PAGE separated protein samples stained with Coomassie blue.
We also characterized a mutant form of Tub4p, Tub4 –34p,
with the mutations F244S and Y247C (Jeng and Stearns,
personal communication). tub4-34 cells are defective in microtubule nucleation and the SPB is deficient in both Tub434p and Spc98p (Marschall et al., 1996). Consistent with the
in vivo phenotypes, we observed that even although expressed in insect cells at the same level as wild-type Tub4p,
Tub4-34p fails to enhance the soluble expression of Spc98p,
suggesting a defective interaction between the two components (Figure 1A). Finally, when coexpressed with GSTSpc110p1–220, only the combination of Spc98p and Tub4p,
but not Spc98p and Tub4-34p, copurifies with GSTSpc110p1–220 (our unpublished data).
Reconstitution and Purification of Tub4p Complex
To further understand the assembly and function of the
three components in the Tub4p complex, we reconstituted
and purified the Tub4p/Spc97p/Spc98p complex expressed
from insect cells by using two methods. In method 1, the
complex was purified using Spc97p-GST (Figure 2A); in
method 2, the complex was purified on the basis of its ability
to bind to GST-Spc110p1–220 (Figure 2B). GST-Spc110p1–220
was subsequently removed by anion exchange chromatography (Figure 2C). Both methods yield complexes containing the three proteins in a stoichiometry of 1 Spc97p:1
Spc98p:2 Tub4p. The minor bands detected by Coomassie
blue staining (Figure 2B) are breakdown products of Spc98p.
Tubulin was not detected in either purified preparation by
Western blot with DM1␣ antibodies, which recognize insect
␣-tubulin (our unpublished data). We also observed that
excess Spc97p was not purified from method 1, consistent
with the fact that stable expression of Spc97p-GST is limited
by the expression of Tub4p. Generally, method 2 produces
about three times more Tub4p complex and has a larger
purification capacity, but method 1 is quicker.
The molecular mass of the Tub4p complex was determined by sucrose gradient centrifugation and gel filtration
chromatography. Purified recombinant Tub4p complex miVol. 13, April 2002
grates predominantly at a peak of 11.6 S in a sucrose gradient (Figure 3B). By gel filtration, the complex has a Stokes
radius of 7.1 nm (Table 2). Based on these values, we estimate the molecular mass of the reconstituted Tub4p complex to be 340 kDa. This number closely matches the predicted molecular mass (300 kDa) of a Tub4p complex based
on the amino acid sequences and stoichiometry of the components.
Reconstitution of Tub4p Complex with Full-Length
Spc110p
Because we were interested in what effects Spc110p has on
the organization and nucleation activity of the Tub4p complex, we reconstituted the Tub4p complex together with
full-length Spc110p. However, unlike GST-Spc110p1–220, the
soluble expression of Spc110p-GST or Spc110p is much
lower making copurification with the Tub4p complex inefficient. We separately purified Spc110p and the Tub4p complex and reconstituted them just before biochemical analyses. One way to increase the solubility of Spc110p-GST
without disrupting the GST/glutathione-Sepharose binding
was to prepare insect cell extract at a higher salt concentration of 300 mM KCl. During purification, the concentration
of salt was reduced to 100 mM KCl to allow association with
the Tub4p complex. Furthermore, we found that coexpressing yeast calmodulin (Cmd1p) with Spc110p-GST greatly
enhances its soluble expression and yield (Figure 4), perhaps
by preventing aggregation. Calmodulin binds the C terminus of Spc110p and in yeast cells, calmodulin is required for
proper assembly of Spc110p multimers (Sundberg et al.,
1996).
Finally, we used a modified GST-copurification assay to
estimate the Kd for binding of purified Spc110p/Cmd1p to
the Tub4p complex. A fixed amount of Tub4p complex was
incubated with different concentrations of Spc110p-GST/
Cmd1p and then glutathione-Sepharose beads were added.
The unbound Tub4p complex was separated from that
1149
D.B.N. Vinh et al.
Figure 4. Yield of Spc110p-GST purified from insect cells is increased by the coexpression of yeast calmodulin, Cmd1p. Western
blot with anti-GST shows expression of Spc110p-GST in the starting
cleared lysates (S), in the insoluble pellets (P), and collected in the
eluates after glutathione-Sepharose beads (E). Loading was equal in
S and P samples and 10-fold more in E. Quantitation of the intensity
of Spc110p-GST bands shows that fivefold more Spc110p-GST is
isolated (E) in the presence of calmodulin.
Characterization of Complex Containing [Spc110p/
Cmd1p ⴙ Tub4p Complex]
Figure 3. Sucrose gradients of reconstituted Tub4p complex and
Spc110p/Cmd1p purified from insect cells. (A) Spc110p/Cmd1p
alone. Asterisk (ⴱ) indicates a breakdown product of Spc110p that
was confirmed with anti-Spc110p Western blot. (B) Tub4p complex.
(C) Tub4p complex ⫹ Spc110p/Cmd1p. In A–C, fractions were
analyzed by SDS-PAGE and the gels stained with Coomassie blue.
Positions of the peak of the tested samples are indicated above each
panel, and positions of internal protein standards are indicated
beneath.
To determine whether Spc110p can organize the Tub4p complex into a higher order structure such as the ␥-TuRC, we
estimated the size of purified Spc110p/Cmd1p alone and a
reconstituted complex containing Spc110p/Cmd1p and the
Tub4p complex. Spc110p/Cmd1p migrates at 4.2 S in a
sucrose gradient, but has a large Stokes radius of 14.3 nm by
gel filtration (Figure 3A and Table 2). Based on these values,
Spc110p/Cmd1p has a molecular mass of 250 kDa and is a
dimer of the Spc110p/Cmd1p complex (predicted molecular
mass based on amino acid sequence is 256 kDa).
To confirm reconstitution of a large complex containing
Spc110p/Cmd1p and Tub4p complex, we tested for comigration of all components in sucrose gradient sedimentation
and gel filtration chromatography. After a short preincubation of purified Spc110p/Cmd1p with the Tub4p complex,
all of Spc110p/Cmd1p was observed to shift from the 4 S
position and cosediment with the Tub4p complex to a peak
of 11.3 S (Figure 3C). This sedimentation coefficient is less
than half that of the ␥-TuRC in higher eukaryotes. Unfortu-
bound to Spc110p-GST/Cmd1p by centrifugation and analyzed by SDS-PAGE. We measured the amount of unbound
Tub4p complex that remains in the supernatant at increasing
concentrations of Spc110p-GST/Cmd1p (Figure 5). Using
this assay, the Kd is estimated to be 150 nM.
Table 2. Physical characterization of Tub4p complex ⫾ Spc110p/
Cmd1p
Spc110p/Cmd1p
Tub4p complex
Spc110p/Cmd1p ⫹
Tub4p complex
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Sedimentation
coefficient (S)
Stokes
radius
(nm)
Estimated
molecular
mass (Da)
4.2
11.6
11.3
14.3
7.1
⬎14.4
250,000
340,000
⬎620,000
Figure 5. Spc110p interacts with the Tub4p complex with modest
affinity. Fitted curve for binding of purified Spc110p/Cmd1p to the
Tub4p complex. Fraction of [unbound Tub4p complex] is drawn as
a function of total [Spc110p-GST/Cmd1p]. Total [Tub4p complex] is
138 nM at all concentrations of Spc110p-GST. The Kd estimated by a
linear regression fit of the data is 150 nM (see MATERIALS AND
METHODS).
Molecular Biology of the Cell
Yeast ␥-Tubulin Complex
nately, we could not determine the Stokes radius of this
large complex as it fractionates in the void volume (⬎14.4
nm) of the gel filtration column (Table 2).
Interactions of Spc110p with Other SPB Core
Components
Electron microscopy studies have indicated that the N- and
C-terminal domains of Spc110p are separated by a coiled
coil rod that acts as a spacer between the central and inner
plaques of the SPB. The C terminus of Spc110p is tightly
attached to the core of the SPB as its N terminus anchors the
Tub4p complex (Kilmartin and Goh, 1996; Sundberg et al.,
1996; Knop and Schiebel, 1997; Nguyen et al., 1998). Current
models propose that the SPB core, consisting of a crystalline
array of Spc42p, acts as a template on which docking proteins such as Spc110p are recruited and assembled (Adams
and Kilmartin, 1999). Extraction of the SPB generates a subcomplex of Spc110p/Cmd1p and two other proteins, Spc29p
and Spc42p (Elliott et al., 1999). However, there have been
conflicting thoughts about whether Spc110p can interact
with both Spc29p and Spc42p or whether Spc29p mediates
the interactions of Spc42p with Spc110p (Adams and
Kilmartin, 1999; Elliott et al., 1999). We are interested in
determining whether the interaction of Spc110p with these
two other core components at the C terminus can affect its
self-assembly and its organization of the Tub4p complex at
the N terminus. We first tested whether Spc110p can bind
both Spc29p and Spc42p by using the GST-copurification
assay with recombinant proteins expressed in insect cells.
For these experiments, we tagged the N terminus of Spc110p
with GST to allow unhindered interactions at the C terminus. GST-Spc110p/Cmd1p interacts independently with either Spc29p-Flag or Spc42p-2PY (Figure 6A). However, the
coexpression of Spc29p-Flag and Spc42p-2PY appears to
hinder the binding of Spc42p-2PY to Spc110p (Figure 6A).
One interpretation of these results is that Spc29p and Spc42p
have an overlapping binding site on Spc110p and neither
mediates the binding of the other to Spc110p. Alternatively,
Spc29p binds to Spc42p and masks the binding site of
Spc42p for Spc110p. However, we have not been able to
detect any significant interaction between Spc29p and
Spc42p when similar copurification experiments were tested
using GST-Spc29p and Spc42p-2PY (GST-copurification) or
Spc29p-Flag and Spc42p-2PY (anti-PY immunoprecipitation) (our unpublished data).
We next tested whether the binding of GST-Spc110p/
Cmd1p to either Spc29p-Flag and/or Spc42p-2PY affects its
ability to bind the Tub4p complex. In a coinfection/copurification experiment, we found that the interaction of Spc29pFlag or Spc42p-2PY to GST-Spc110p/Cmd1p has little effect
on its ability to bind to the Tub4p complex (Figure 6B).
In Vitro Microtubule Nucleation Activity of Tub4p
Complex
Although soluble Tub4p exists predominantly in stable
Tub4p complexes, microtubules are only observed to emanate from the SPB. One hypothesis is that the microtubule
nucleation capacity of the cytoplasmic Tub4p complex is
minimal until it is recruited to the SPB by a protein such as
Spc110p. We therefore tested whether the reconstituted
Tub4p complex has microtubule nucleation activity and
Vol. 13, April 2002
Figure 6. Spc110p/Cmd1p can interact with Spc42p or Spc29p
independently of each other. (A) GST-Spc110p/Cmd1p was coexpressed in insect cells with either Spc42p-2PY or Spc29p-FLAG or
both. S is the starting cleared lysate and E is the eluate from
glutathione-Sepharose beads. Protein samples were analyzed by
Western blot with anti-GST, anti-PY, and anti-FLAG antibodies. The
lower migrating band detected by anti-GST is a GST-Spc110p breakdown. (B) Interactions at the C terminus of Spc110p with Spc42p or
Spc29p do not seem to affect its ability to interact with the Tub4p
complex. Insect cells were infected with different combinations of
viruses as indicated, and the Tub4p complex was tested for its
binding to GST-Spc110p/Cmd1p. Note that the Tub4p complex is
present at a 10-fold molar excess to GST-Spc110p/Cmd1p in each
cleared crude lysate (our unpublished data). Eluates were analyzed
by Western blot.
whether interacting with purified Spc110p enhances this
activity. To measure nucleation, we used a modified solution assay adapted from Oegema et al. (1999). Rhodaminelabeled bovine tubulin was incubated with recombinant
Tub4p complex or buffer alone, and the resulting polymerized microtubules were pelleted onto coverslips and
counted using fluorescence microscopy (Figure 7A). Because
there is some variability, we report the average of all experiments and also show the highest and lowest values. An
average of six independent experiments shows that incubating 2.0 mg/ml tubulin with 50 nM Tub4p complex promotes
microtubule polymerization about threefold above the background level of a buffer control (Figure 7B) (a one-sided
Wilcoxon rank sum test yielded a P value of 0.021). A
fourfold increase in the concentration of Tub4p complex
increased activity by 40% (our unpublished data). Tub4p
complexes obtained by either purification method 1
1151
D.B.N. Vinh et al.
(Spc97p-GST) or method 2 (GST-Spc110p1–220), which has an
additional purification step, gave similar results. Thus, the
microtubule-nucleating activity observed is likely due to the
reconstituted Tub4p complex. The addition of Spc110p has
no effect because 50 nM Tub4p complex preincubated with
350 nM Spc110p/Cmd1p produced a similar level of activity
as Tub4p complex alone (Figure 7B). Finally, the nucleation
activity of the reconstituted Tub4p complex was the same
whether purified yeast tubulin or bovine tubulin was used
(our unpublished data).
Because of the low nucleating activity, we tested the binding of the Tub4p complex to microtubules by using a microtubule copelleting assay. The Tub4p complex cosediments
with taxol-stabilized microtubules (Figure 7C). This binding
is specific because incubating the negative control bacterial
GST with taxol-microtubules does not pellet a significant
amount of GST (Figure 7C).
Sedimentation of Tub4p Complex from Yeast Whole
Cell Extract
The ␥-tubulin complex of higher eukaryotic cells contains
additional components that enhance the nucleating activity
of the small complex (Oegema et al., 1999). No evidence
exists for such a large complex in yeast. Furthermore, results
from our studies of recombinant Tub4p complex and
Spc110p/Cmd1p indicate a low microtubule-nucleating activity. We sought to compare the recombinant Tub4p complex to the native complex in yeast cells. The sedimentation
coefficient of the recombinant Tub4p complex is identical to
that isolated from yeast (11.6 S) when sedimented in a
Tris-based buffer (TB100) developed by Knop and coworkers (Figure 8A; our unpublished data). However, because
previous experiments have been performed with a buffer
that may interfere with microtubule polymerization, we
sedimented yeast extracts by using buffer HB100 known to
support microtubule nucleation. Interestingly, we found
that under these conditions, a significant portion of Tub4p
migrates at 22 S (Figure 8, B and C). High salt does not
dissociate this large complex because a cell extract prepared
in HB plus 400 mM KCl still retains the 22 S peak (our
unpublished data). The 22 S Tub4p complex does not migrate with endogenous Spc110p, which has a sedimentation
coefficient of 4.4 S under all conditions (Figure 8C). This
result is consistent with the fact that most Spc110p (ⱖ75%;
Figure 7. Reconstituted Tub4p complex can promote microtubule
polymerization at a low level. (A) Representative video fields of
rhodamine-labeled microtubules from the nucleation assay polymerized in the presence of buffer or recombinant Tub4p complex.
Bar, 10 ␮m. (B) Summary of the nucleation assay. For each experiment, the number of microtubules is counted from 20 fields and an
1152
average number of microtubules/field is taken. The indicated #Microtubules/field is normalized as the total number of microtubules
per field that would have been counted in a 5-␮l reaction. For each
sample, an average (of 3– 6 independent experiments), a high and a
low result are indicated. For Tub4p complex alone, the average was
taken of results from two experiments by using complexes purified
by method 1 and 4 experiments with complexes purified by method
2. We estimate that the concentration of microtubules produced in
the presence of 50 nM Tub4p complex is 1.8 pM. (C) Reconstituted
Tub4p complex was tested for its ability to bind taxol-stabilized
microtubules in a copelleting assay. After pelleting the microtubules, the supernatant and pellet were separated and both analyzed
by Western blot to look for components of the Tub4p complex or the
negative control, bacterial GST. Tubulin pellets were also analyzed
by Ponceau S staining of the Western blot. In this experiment, 15 nM
Tub4p complex was incubated with 11.2 ␮M polymeric tubulin
containing 3 nM microtubule ends.
Molecular Biology of the Cell
Yeast ␥-Tubulin Complex
Figure 8. Identification of the 22 S Tub4p complex
from yeast extract. (A and B) A logarithmic culture
of cells containing SPC97-3HA cells was divided into
two and lysed with either TB100 (A) or HB100 buffer
(B). (C) Wild-type yeast cell extract prepared in
HB100. Cleared cell extracts were loaded onto 10 –
40% sucrose gradients prepared in the same buffer
as the cell extract. Fractions (P is the pellet) were
TCA precipitated and analyzed by Western blot
with anti-HA, anti-Tub4p, or anti-Spc110p antibodies. Internal protein standards were included in each
gradient and were analyzed directly by Coomassie
stained SDS-PAGE without TCA precipitation. The
estimated sedimentation coefficients of the Spc973HA/Tub4p complexes are marked above each gradient.
our unpublished data) is found in the SPB pellet, and soluble
Spc110p is not associated with Tub4p complexes, perhaps
due to the modest affinity of Spc110p for the Tub4p complex.
Note that the recombinant form of Spc110p/Cmd1p has the
same sedimentation coefficient as that derived from yeast
(compare Figure 3A with 8C).
We determined whether the 22 S Tub4p complex includes
other components of the small Tub4p complex by analyzing
cell extract from a yeast strain harboring a single genomic
copy of SPC97-3HA. The majority of Spc97p-3HA migrates
with Tub4p at 22 S in a sucrose gradient (Figure 8B), suggesting that the large Tub4p complex contains Spc97p. The
fact that Spc97p-3HA sediments predominantly at 22 S indicates that the fraction of Tub4p that sediments at smaller
S values are not associated with Spc97p. ␣-Tubulin (analyzed by Western blot with YOL/34 antibody) was not
associated with the 22 S peak (our unpublished data).
The size of the large Tub4p complex resembles that of
vertebrate ␥-TuRCs (25–35 S). The ␥-TuRC is composed of
many small ␥-TuSCs and various accessory proteins that are
believed to link multimers of ␥-TuSCs and enhance their
nucleating activity (Oegema et al., 1999; Moritz et al., 2000;
Zhang et al., 2000). To determine whether the 22 S Tub4p
complex contains more than one small Tub4p complex, we
tested for physical interactions between two different tagged
versions of Spc97p. A heterozygous yeast strain was constructed to contain a genomic copy of SPC97-3HA and a
Vol. 13, April 2002
genomic copy of SPC97-13MYC. Cell extract was prepared
in HB100 and analyzed by sucrose gradient centrifugation.
All three Spc97p-13MYC, Spc97p-3HA, and Tub4p cosediment at a peak of ⬃23 S (Figure 9A). A fraction of Spc97p13MYC and Spc97p-3HA also sediments at lower S values,
suggesting the presence of smaller tagged Spc97p complexes. To demonstrate that the two tagged versions of
Spc97p were in the same complex, we immunoprecipitated
the same cell extract with anti-HA or anti-MYC antibodies.
Anti-MYC antibodies precipitate both Spc97p-13MYC and
Spc97p-3HA and anti-HA antibodies precipitate both
Spc97p-3HA and Spc97p-13MYC (Figure 9B). In both cases,
Tub4p was also coprecipitated, suggesting association between complexes containing Spc97p and Tub4p. These coprecipitations are specific because subjecting an extract from
SPC97-3HA cells with anti-MYC antibody does not bring
down Spc97p-3HA or Tub4p (Figure 9B). We conclude that
at least a subset of the 23 S Tub4p complexes found in
SPC97-3HA/SPC97-13MYC cells contain more than one
Spc97p.
DISCUSSION
Orthologs of components of the yeast Tub4p complex make
up the ␥-TuSC, which is the core unit of vertebrate ␥-TuRCs.
The fact that no complex similar to the ␥-TuRC had been
1153
D.B.N. Vinh et al.
Knop and Schiebel, 1997), this is the first time the stoichiometry has been determined. This stoichiometry is identical
to that of the Drosophila ␥-TuSC (Oegema et al., 1999), suggesting conserved assembly, in addition to composition, of
core components of the microtubule nucleation machinery.
The native Tub4p complex derived from yeast extract
sediments at 11.6 or 22 S, depending on the buffers used to
prepare the extract (see below). In yeast extract prepared in
a Tris buffer, TB100, the 11.6 S complex predominates. The
recombinant complex prepared from insect cells that consists of one molecule of Spc98p, one molecule of Spc97p, and
two molecules of Tub4p also sediments at 11.6 S. This sedimentation coefficient conflicts with that of Knop et al. (1997)
who report that the Tub4p complex isolated from yeast in
TB-100 sediments at 6 S. However, because the native Tub4p
complex in their studies migrates immediately below the
protein standard ␤-amylase, which has a sedimentation coefficient of 8.9 S (Sober, 1968), the actual value appears to be
closer to 11.6 S.
Interactions between Tub4p Complex and Spc110p
Figure 9. The large Tub4p-complex contains more than one
Spc97p. (A) Sucrose gradient of cell extract prepared from strain
SPC97-13MYC/SPC97-3HA in HB100 buffer. Fractions were TCA
precipitated and analyzed by Western blots with anti-MYC, antiHA, or anti-Tub4p antibodies. Note the comigration of Spc97p13MYC, Spc97p-3HA, and Tub4p at 23 S. (B) Immunoprecipitations
of cell extracts from strain SPC97-13MYC/SPC97-3HA or SPC973HA by using anti-MYC or anti-HA antibodies. Specific proteins
were identified by Western blot. For each immunoprecipitation, S is
the starting cleared lysate, and B is the fraction solubilized from the
immunoprecipitate–protein-G beads. S1 and S2 are different sources
of cell extracts.
identified in S. cerevisiae raised the question of whether the
mechanism of microtubule nucleation and its regulation in
yeast are simplified versions of those in higher eukaryotes.
To further understand the assembly and function of the
yeast ␥-TuSC, we characterized the Tub4p complex alone
and when bound to the SPB docking protein Spc110p by
using recombinant proteins expressed in insect cells. The
strong similarities we found between the small Tub4p complex and the ␥-TuSC in other organisms led us to reexamine
the presence of a large Tub4p complex in yeast. We present
herein evidence for a large Tub4p complex from yeast extracts that may contain multimers of small Tub4p complexes.
Reconstitution of Tub4p Complex
We reconstituted the small Tub4p complex from insect cells
to produce the large amount of purified complexes required
for biochemical analyses. The molecular mass of the reconstituted Tub4p complex is 340 kDa, which closely matches
the molecular mass based on the measured stoichiometry of
1 Spc98p:1 Spc97p:2 Tub4p in our purified complexes. Although previous immunoprecipitation studies identified the
three components of the Tub4p complex (Knop et al., 1997;
1154
We and others have previously identified Spc110p as the
docking protein of the Tub4p complex to the nuclear side of
the SPB (Knop and Schiebel, 1997; Nguyen et al., 1998). The
recent identification of kendrin/pericentrin as the mammalian ortholog of Spc110p suggests a universal mechanism of
recruitment or attachment of ␥-tubulin complexes to the
centrosome (Flory et al., 2000). In these studies, we have
reconstituted the Tub4p complex with Spc110p to determine
what effects Spc110p has on the organization and nucleation
activity of Tub4p complexes.
By the two-hybrid assay, we previously observed diverse
interactions between the components of the Tub4p complex
and Spc110p (Nguyen et al., 1998). To test these interactions
in another way and to avoid the background interference of
endogenous yeast proteins in the two-hybrid assay, we characterized the interactions of proteins expressed and purified
from insect cells. Confirming our two-hybrid results, we
observed that Spc98p is the primary component of the
Tub4p complex that binds to the N terminus of Spc110p. The
affinity of Spc98p for Spc110p and the fact that Spc98p is the
only component of the Tub4p complex with a nuclear localization signal may be important in regulating the recruitment of the Tub4p complex to the SPB nuclear side. Unexpectedly, we found that complexing with Tub4p enhances
the expression of either soluble Spc98p or Spc97p. One
would predict that free Tub4p, but not free Spc98p or
Spc97p, can exist in cells. In fact, yeast extracts typically
contain species of Tub4p with small sedimentation coefficients that do not cosediment with Spc97p, whereas all of the
Spc97p cosediments with Tub4p.
We were particularly interested in characterizing fulllength Spc110p and its interactions with the Tub4p complex.
We first discovered that the solubility of Spc110p in insect
cells is much improved when yeast calmodulin or Cmd1p is
coexpressed. This result is consistent with the phenotypes of
mutants harboring an Spc110p defective in binding Cmd1p
(Sundberg et al., 1996). At the nonpermissive temperature,
spc110-220 cells accumulate a nuclear aggregate of Spc110p
that is detached from the SPB and devoid of Cmd1p, suggesting that Cmd1p promotes the proper assembly of
Spc110p required for incorporation into the SPB.
Molecular Biology of the Cell
Yeast ␥-Tubulin Complex
From its hydrodynamic characteristics, purified recombinant Spc110p/Cmd1p is estimated to have two molecules
each of Spc110p and Cmd1p and a molecular mass of 250
kDa. A small measured sedimentation coefficient in combination with the large Stokes radius suggests an asymmetrical structure consistent with the rod shape of its extensive
coiled coil region (Kilmartin et al., 1993). The binding of
Spc110p/Cmd1p to the Tub4p complex generates a larger
complex with a sedimentation coefficient of 11.3 S, but not as
high as the 25–32 S values for vertebrate ␥-TuRCs (Stearns
and Kirschner, 1994; Meads and Schroer, 1995; Murphy et al.,
1998; Oegema et al., 1999). Because Spc110p/Cmd1p plus
Tub4p complex together migrate in the void volume of a gel
filtration column, we cannot determine the molecular mass
of this larger complex. Soluble Spc110p/Cmd1p in yeast
extracts sediments at 4.2 S identical to that of the recombinant Spc110p/Cmd1p. The native soluble Spc110p is not
associated with the Tub4p complex consistent with the low
abundance of these proteins and the moderate Kd (150 nM)
for their binding.
We tested whether Spc29p or Spc42p enhances the affinity
of Spc110p for the Tub4p complex. Both Spc29p and Spc42p
are core components required for SPB duplication (Donaldson and Kilmartin, 1996; Elliott et al., 1999) and are part of a
template that recruits and organizes the assembly of
Spc110p onto the central plaque of the SPB (Adams and
Kilmartin, 1999). We provide evidence herein that Spc110p/
Cmd1p binds directly to both Spc42p and Spc29p. However,
we found that the binding of Spc110p to either Spc42p
and/or Spc29p does not affect its interactions with the
Tub4p complex. Interestingly, recombinant Spc42p and
Spc29p do not mediate the binding of each other to Spc110p;
instead, their binding appears to be competitive. How
Spc110p, calmodulin, Spc29p, and Spc42p assemble into the
central plaque remains an open question.
Microtubule Nucleation and Binding by Tub4p
Complex
Studies in Drosophila indicate that the ␥-TuRC has robust
microtubule nucleation activity, but the individual ␥-TuSC
subunit exhibits only weak activity (Oegema et al., 1999).
Because yeast cells were not known to possess a large Tub4p
complex analogous to the ␥-TuRC, we tested whether microtubule polymerization in yeast requires only the small
Tub4p complex or depends on a critical interaction between
the Tub4p complex and the SPB. Recruitment to the SPB
may result in the organization of Tub4p complexes into a
structure that has robust nucleation activity similar to the
␥-TuRC. Furthermore, although the small Tub4p complex
can interact directly with the SPB via a docking protein such
as Spc110p, the vertebrate ␥-TuSC requires additional components contained in the ␥-TuRC (Moritz et al., 1998; Zhang
et al., 2000). We measured the nucleation activity of the
reconstituted Tub4p complex alone and when it is bound to
Spc110p. Our results indicate that the Tub4p complex has a
low intrinsic ability to promote microtubule polymerization
and interaction with Spc110p does not alter this activity.
Although we cannot completely rule out the presence of
insect cell contaminants in our preparations, we attribute the
low nucleation activity to the reconstituted Tub4p complex
for several reasons. First, an additional purification step
does not alter the nucleation activity of the Tub4p complex.
Vol. 13, April 2002
Second, we did not find any trace of insect tubulin contaminants. Finally, this low nucleation activity is equivalent to
the activity reported for Drosophila ␥-TuSC extracted from
the ␥-TuRC (Oegema et al., 1999) and more recently, reconstituted from Sf9 insect cells (Gunawardane et al., 2000).
A Larger Tub4p Complex in Yeast
Our findings that the Tub4p complex with or without
Spc110p/Cmd1p only has low nucleation activity suggested
to us that components associated with an active Tub4p
complex are missing. Because the ends of yeast microtubules
are capped similarly to microtubules nucleated in the presence of vertebrate ␥-TuRC, it is possible that other as yet
unknown yeast proteins may organize the Tub4p complexes
into a multimeric ring structure such as the ␥-TuRC. Careful
analysis of yeast extracts prepared under conditions known
to promote microtubule nucleation identified a stable 22 S
complex containing Tub4p and Spc97p. The size of this
complex is close to the 25–32 S size of vertebrate ␥-TuRCs.
This complex is not associated with Spc110p or tubulin and
contains more than one molecule of Spc97p, similar to the
␥-TuRC. Future analyses will identify the components of the
complex and its role in microtubule nucleation.
ACKNOWLEDGMENTS
We thank R. Jeng and T. Stearns for antibodies and plasmids; R.
Deshaies for cell line AK1310; K. Kaplan and P. Sorger for plasmids
and advice; and M. Gupta and R. Himes for yeast tubulin. We thank
L. Wordeman and S. Francis for helpful discussions and comments
on the manuscript and A. Hunter for technical advice. This work
was supported by National Institutes of Health grant R01 GM40506, National Institute of General Medical Sciences (to T.N.D.).
D.V. was supported by Public Health Service National Research
Service Award F32 GM-17945, National Institute of General Medical
Sciences.
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